Effect of Honey and Aqueous Garlic Extracts against Short-Term Exposure of Cigarette Tobacco Smoking in Mice: Histopathological and Biochemical Investigations

It is well known that cigarette smoking adversely affects human health and induces oxidative stress in most vital organs. This study aims to assess the biochemical, histological, and ultrastructural values of honey and garlic extracts in ameliorating the effects of short-term exposure to cigarette smoke in mice. Forty-eight mice were randomly divided into six equal groups: group I was exposed to fresh air only, group II was exposed to cigarette smoke, group III was given 0.2 ml of honey extract, group IV was exposed to cigarette smoke and was given 0.2 ml of honey extract, group V was given 0.2 ml of garlic extract, and group VI was exposed to cigarette smoke and was given 0.2 ml of aqueous garlic extract. These exposures were repeated daily for 21 consecutive days among the treated groups. By the end of the third week, the animals were euthanized by physical cervical dislocation. Blood was taken for biochemical study, and the selected organs of the liver, kidney, and jejunum were processed for histological and ultrastructural studies. The biochemical results showed that short-term exposure of experimental mice to cigarette smoking did not alter the liver function tests except for decreasing the albumin level. Moreover, cigarette smoking elevates the concentration of carbonyl protein content and cystatin C. Histologically, the use of honey and garlic showed good protection to the liver, kidney, and jejunum, which was proved by transmission electron microscopy, in addition to lowering the oxidative stress biomarkers. In conclusion, using honey and/or garlic helps protect the liver, kidney, and jejunum against the hazardous effects of cigarette smoke.


Introduction
Tobacco smoking through cigarettes or waterpipes contains multiple toxicants such as transition metals, carbon monoxide, aldehydes, nicotine, nitrosamine, and solid particulate matter [1].Te gaseous phase of cigarette smoke and the aqueous phase of cigarette tar contain signifcant amounts of free radicals with reactive oxygen and nitrogen species which eventually promote the production of hydroxyl radicals [2].Tese chemical toxicants induce oxidative stress, vasoconstriction, infammation, DNA damage, and adverse epigenetic regulation that contribute to organ dysfunction associated with adverse clinical outcomes [1][2][3].
Furthermore, prolonged secondhand exposure to tobacco smoking could progress into respiratory and cardiovascular risks in parallel with chronic obstructive pulmonary diseases [4].
Natural antioxidants are considered excellent therapeutic remedies to decrease oxidative stress by direct scavenging of reactive oxygen and nitrogen species that could afect human health [5].Antioxidants are chemicals that are able to lower the activity of free radicals, therefore attenuating the risk of oxidative stress, and preserve the cellular components from alterations or damage [6].
Tis work aims to assess the ameliorative efect of the aqueous extracts of garlic and honey against secondhand exposure to cigarette tobacco smoking using histopathological and clinical chemistry parameters to compare among the experimental groups of albino mice.

Preparation of Aqueous Garlic and Honey Extract.
Bee honey and garlic were freshly purchased from the local market.Both aqueous garlic and honey extracts were prepared using the procedure described by Waheeb and Ali [13].

Animal Grouping and Dose
Administration.Tis study was operated according to the instructions of the Scientifc Committee in the School of Science at the University of Jordan in terms of animal handling and care.Tese instructions are consistent with the NIH Guide for the Care and Use of Laboratory Animals 2011 [14].Male albino BALB/c mice were purchased from the Animal Household/ University of Jordan.All adult males were about eight weeks old, weight 22 ± 3 g.Te cigarettes: red labeled (L&M; 10 mg tar, 0.8 mg nicotine, and 10 mg CO) marked cigarettes were purchased from local markets (Philip Morris, Jordan).
Forty-eight adult male mice were randomly divided into 6 experimental groups: each group contains 8 mice.Group I (control): mice were exposed to fresh air only.Group II (SMK): mice were exposed to cigarette smoking.Group III (honey): mice were given 0.2 ml of prepared aqueous honey extract through oral gavage.Group IV (SMK + honey): mice were exposed to cigarette smoking and given 0.2 ml of prepared aqueous honey extract through oral gavage.Group V (garlic): mice were given 0.2 ml of prepared aqueous garlic extract through oral gavage.Group VI (SMK + garlic): mice were exposed to cigarette smoking and given 0.2 ml of prepared aqueous garlic extract through oral gavage.Tese exposures were repeated daily for 21 consecutive days among the treated groups.
Te process of cigarette smoking exposure was explained by Alzbeede et al. [15] using a modifed smoking machine described by Shraideh et al. [16].

Tissue Separation and Processing
. By the end of the 3rd week, the mice were euthanized by physical cervical dislocation.Blood was withdrawn through inserting a capillary tube into the venous orbital plexus, and serum was separated from the blood (clotting time 60 minutes in room temperature under dark conditions, followed by centrifugation 3800 rpm-10 minutes) for biochemical analysis.
Mice were dissected to obtain the liver, kidney, and jejunum.Tese organs were washed with phosphate bufered saline (PBS) (0.01 M-pH 7.2) and fxed in 10% formal saline for light microscopy or 2.5% glutaraldehyde in PBS for electron microscopy techniques.Steps of light and transmission electron microscopy were described by Alzbeede et al. [15].Organs were dehydrated with ascending grades of ethanol, cleared with xylene, and impregnated and embedded in melted parafn wax for the light microscopy technique.Ten, these samples were sectioned at 5 μm thickness, stained with hematoxylin and eosin stains, mounted by DPX, and examined using light microscopy.
Besides, small pieces of the selected organs were postfxed with 1% osmium tetroxide, dehydrated with ethanol, cleared by propylene oxide, and infltrated with 1 : 1 propylene oxide: resin, and embedded in epoxy resin.Tese specimens were sectioned using an ultramicrotome, then mounted on copper grids and stained with uranyl acetate and counter-stained with lead citrate, and then, the sections were then ready to be examined by transmission electron microscopy.

Biochemical Testing.
Te separated serum was used to estimate some biochemical markers such as liver function tests, e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), gammaglutamyl transferase (cGT), total protein, albumin, globulin, total bilirubin, and kidney function tests, e.g., creatinine, urea, and cystatin C. Besides, quantitative measurements of carbonyl protein content and malonaldehyde (MDA) were performed to evaluate protein and lipid peroxidation, respectively.All biochemical tests listed above were completed using a fully automated biochemical analyzers (HumaStar 600, Human Diagnostics Worldwide, Germany) except cystatin C and carbonyl protein contents that were quantifed according to the instructions provided by the kit manufacturer (ab119590, Abcam, UK) and (ab126287, Abcam, UK), respectively, using a 96-well plate reader (Synergy HTX, BioTeK, USA).Te evaluation of lipid peroxidation was performed by the determination of malondialdehyde (MDA) concentration in plasma samples, according to Lima et al. [17].A serum sample of 250 μl was mixed with 500 μl of 0.6% 2-thiobarbaturic acid (TBA) (T5500, Sigma Aldrich, USA).

Statistical Analysis. Te results were tabulated in
GraphPad Prism version 8.2.1, IBM, USA, for statistical analysis.All data of the treatment groups for each biochemical test were analyzed by one-way ANOVA, followed by a Tukey post hoc comparison test and considering P value <0.05.

Biochemical Investigation of Liver, Kidney, and Oxidative
Stress Biomarkers.Mice exposed to cigarette smoke showed a signifcant decrease in albumin concentration compared to the control, honey, and garlic groups at P < 0.01 (0.0073), P < 0.01 (0.0016), and P < 0.05 (0.0158), respectively (Table 1).However, treatments with honey or garlic were in parallel with exposure to cigarette smoke did not show any signifcant diferences with control, honey, and garlic groups at P < 0.05 (Table 1).Te AST activity result revealed that the smoking group has slightly increased with a signifcant diference against the garlic group only P < 0.05 (0.0353) (Table 1).Otherwise, there are no signifcant diferences among the experimental groups (Table 1).Te concentrations of total protein, globulins, total bilirubin, and the enzymatic activity of ALT and cGT did not show any signifcant diferences among the treated groups at P < 0.05 (Table 1).
Kidney function tests illustrated that the concentrations of creatinine and urea did not show any signifcant diferences among the study groups (Table 1).However, the cystatin C levels of the SMK group showed signifcant results compared to honey and garlic groups at P < 0.01 (0.0073) and P < 0.01 (0.0016), respectively, but this slight increase in cystatin C level was not signifcant against the control group at P < 0.05 (Table 1).
Carbonyl protein content is illustrated in Figure 1(a) and the SMK group has signifcant diferences with all other experimental groups at diferent P values (P < 0.0001 versus control, honey, and garlic groups, respectively) and (P � 0.0006 versus SMK + honey; P � 0.0021 versus SMK + garlic group).SMK + honey group did not show any signifcant diferences with the control or honey group.Te same thing was revealed with the garlic group that did not show any signifcant diferences with the control and garlic group at P < 0.05.
Lipid peroxidation level was estimated depending on malonaldehyde quantifcation (Figure 1(b)).It showed that the SMK group has a signifcantly higher concentration than the control, honey, and garlic groups at P < 0.0001.Moreover, the SMK group showed signifcant diferences with SMK + honey (P � 0.0006) and SMK + garlic (P � 0.0021).Te SMK + honey group did not show any signifcant differences with the control or honey group.Te same thing was revealed with the garlic group that did not show any signifcant diferences with the control and garlic group at P < 0.05.

Histopathological Examination of Hepatic Cells Using
Light Microscopy.Sections of the liver of the control group had shown histologically normal liver architecture, containing normal hepatocytes and sinusoids, a central vein, and many binucleated hepatocytes (Figure 2(a)).Te histological sections of the liver of the CS group showed that Kupfer cells became prominent and increased in number and sinusoids are dilated (Figure 2(b)).Hepatocytes showed a high degree of vacuolization, chromatin of nuclei was condensed into peripheral clumps and congested blood vessels (Figure 2(b)).Following treatment with honey, the liver of mice of this group showed signs of partial recovery (Figure 2(c)).Te treatment resulted in improvement in hepatocyte architecture, less vacuolization, and less infltration of immune cells and Kupfer cells.In addition, treatment with garlic showed a certain degree of protection against CS damage (Figure 2(d)).Te hepatocytes are still showing a lesser degree of vacuolization and less degree of infltration of Kupfer cells in comparison to the CS group (Figure 2(d)).

Histopathological Examination of Nephrons Using Light
Microscopy.Te section of kidney samples from the control group is represented in Figure 3(a).It showed normal integrity of the renal cortex, with a normal renal corpuscle characterized by glomeruli and Bowman's capsule surrounded by proximal and distal convoluted tubules (Figure 3(a)).
Te kidney sections of mice exposed to cigarette smoking showed several abnormalities, including widening of the renal space, mild condensed glomeruli, heterochromatic nuclei, cell vacuolization, partially disrupted epithelia, or even desquamation of the tubular cells.Following treatment with honey, the kidney of mice of this group showed signs of partial prophylaxis by demonstrating improvements in renal architecture, condensation of the glomerulus, no cellular vacuolization, and normal nuclei of tubular cells (Figure 3(c)).Treatment with garlic showed a certain degree of protection of the kidney against CS damage (Figure 3(d)).Tere is still less glomeruli condensation and partial disruption of the tubular epithelium (Figure 3(d)).

Histopathological Examination of the Jejunum Using Light
Microscopy.Te cross-section of the jejunum of the control mice revealed the typical architecture of the epithelial layer of the small intestine associated with normal enterocytes, goblet cells, and intestinal glands with Paneth cells (Figure 4(a)).Exposure to cigarette smoking showed several abnormalities as enterocytes appeared with heterochromatic nuclei and cellular vacuolization, in addition to increasing the number of Paneth cells and infltration of macrophages in the lamina propria (Figure 4(b)).Following treatment with honey, the jejunum of mice of this group showed good protection against CS; there is an improvement in intestine architecture, no cellular vacuolization, and normal nuclei of enterocytes (Figure 4(c)).Moreover, treatment with garlic ameliorated the efect of CS; the intestinal architecture appears normal, cellular vacuolization is less, and normal nuclei of enterocytes (Figure 4(d)).

Ultrastructural Observation of Hepatic Cells Using Transmission Electron Microscopy.
In ultrathin sections of the liver from the control experimental mice, normal hepatocytes were seen (Figure 5(a)).Tese cells had characteristically large, round, predominantly euchromatic nuclei.Tere were several obvious, clearly cristae-containing mitochondria in the cytoplasm, as well as many profles of rough endoplasmic reticulum (Figure 5(a)).In contrast, hepatocytes of mice that were exposed to cigarette smoking displayed polymorphic mitochondria and mild deterioration in the cristae and matrix; the nuclei contained some chromatin condensation next to the inner margins of the nuclear envelope; the nuclear envelope appeared irregular in shape; a rough endoplasmic reticulum appeared scattered with dilated cisternae; and the cytosol content was distorted with the presence of some vacuolization (Figure 5(b)).
Comparing portions of hepatocytes treated with honey to hepatocytes that had been exposed to smoke, the outcomes were better.It revealed decreased cellular blebbing, preserved normal mitochondrial shape and size, and increased euchromatic chromatin proportions in the nuclei (Figure 5(c)).Ultrastructural examination of the liver in mice receiving the aqueous garlic extract showed good protection against CS, as seen in Figure 5(d).Te mitochondria appear to have proper cristae, and the cells did not exhibit vacuolization or multilamellar structures.In addition, chromatin is less condensed in nuclei (Figure 5(d)).

Ultrastructural Observation of Nephron Using Transmission Electron Microscopy.
Te ultrathin section from the cortical part of the nephron in the control group showed normal integrity of the renal cortex, with normal glomeruli surrounded by proximal and distal convoluted tubules (Figure 6(a)).Te examined ultrathin sections of cigarette smoke exposed showed a high degree of cytoplasmic vacuolization and polymorphic, swollen mitochondria.

Journal of Toxicology
Tere was also extruded material in the renal space and partially disrupted capillary endothelium (Figure 6(b)).Following treatment with honey, the kidney of mice of this group showed good protection against CS (Figure 6(c)).Te treatment resulted in improvement in kidney ultrastructure, a smaller number of vacuoles and lysosomes in the cytoplasm, and mitochondria looking with normal cristae.Moreover, nuclei have less condensed chromatin surrounded by a nuclear envelope with an intact appearance (Figure 6(c)).Following treatment with garlic, the kidney of mice of this group showed good protection against CS (Figure 6(d)).Renal corpuscle has normal architecture, no vacuolization of cells, normal fltration membrane, and intact endothelium.Podocytes have euchromatic nuclei (Figure 6(d)).

Ultrastructural Observation of the Jejunum Using
Transmission Electron Microscopy.Te jejunum of the control group showed normal architecture: enterocytes with densely packed apical microvilli, mitochondria with normal cristae, and intercellular junctions; all cellular organelles were enclosed within well intact and resembled plasma membranes with deformities.Te ultrastructural examination of the jejunum in mice exposed to cigarette smoking showed to exhibit edema, minor deformation, and dilated cell membranes that resembled blebs (Figure 7(b)).Tey have excessively thick bodies with tiny, lipid-containing vacuoles dispersed throughout the cytoplasm, spherical mitochondria with damaged cristae, and uneven, shortened microvilli baselines (Figure 7(b)).Due to cellular swelling, the enterocytes in this group partially lost their sinuosity, a sign of moderate to severe structural damage (Figure 7(b)).
Following treatment with honey, the jejunum of mice of this group showed good protection against CS (Figure 7(c)).Te treatment resulted in improvements in enterocyte ultrastructure.No cellular vacuolization and euchromatic nuclei of enterocytes (Figure 7(c)).Following treatment with garlic, the jejunum of mice of this group showed good protection against CS (Figure 7(d)).Te treatment resulted in improvements in intestine ultrastructure, no cellular vacuolization, and euchromatic nuclei of enterocytes.Te goblet cells showed a normal appearance with extracellular secretions (Figure 7(d)).

Discussion
Despite recent declines in its prevalence, tobacco smoking remains among the primary causes of illness and early death globally [18].Tobacco smoking in all its forms has harmful efects on active and passive smokers, leading to oxidative stress due to chemical constituents containing various free radicals [19].Te biochemical results of this study showed that short-term exposure of experimental mice to cigarette smoking did not alter the liver function tests except for a decrease in the albumin level.Tese results are inconsistent with those of Nemmar et al. that showed a signifcant increase in the enzymatic activity of ALT and cGT by the action of passive inhaled cigarette smoking in mice [20].
Te assumption of a decline in the albumin level caused by severe hepatic damage that could have a direct efect on albumin production or leakage of albumin through renal fltration were negligible since most markers of liver and kidney function tests were normal in this study.Moreover, according to a case-control study of patients with acute aortic dissection, hypoalbuminemia, smoking, and hypertension are strongly associated with this condition [21].Another fnding of this study is that short-term inhaled cigarette smoking afected the albumin and cystatin C concentrations in serum, which were corrected by oral administration of aqueous honey or garlic extract.In addition, aqueous extracts of honey or garlic given orally have antioxidative capabilities since they reduce the levels of lipid peroxidation and protein carboxylation biomarkers in the serum of the experimental mice used in this investigation.
Histologically, it is apparent that the use of honey and the aqueous solution of garlic had a protective efect on the tissues studied; hepatocytes appeared normal, kidney tissue showed almost complete recovery, and the enterocytes of the jejunum appeared normal.Tis is consistent with the biochemical fndings.Cigarette smoking caused abnormal physiological indices (such as reduced body weight, blood lipid levels, and food intake) that resulted in hepatocellular damage, abnormalities in the liver transcriptome during lipid metabolism, and disruption of the gut microbiota in mice [22][23][24].Moreover, other fndings showed the renal function of mice was negatively afected by exposure to cigarette smoking, which led to an acceleration of renal fbrosis and cystic development as well as a physiological reduction in glomerulus fltration rate [25,26].Exposure to cigarette smoke increases intestinal mucosal disruption, intestinal barrier dysfunction, oxidative stress, apoptosis, and tight junction component dysregulation [27][28][29].It also causes mucosal infammation with specifc changes in Paneth cell granules [30].
In this study, parallel treatment with honey or aqueous garlic extract showed signs of partial recovery in hepatocyte architecture, slight improvement in cellular compartments in the cortex part of the kidney, and maintain the morphological structure of the intestinal mucosal layer against passive exposure to cigarette smoking.
It was explained that garlic extracts containing active compounds such as allicin, diallyl disulfde, disulfde, and S-allylcysteine have antihypertensive, inhibiting the assembly and disassembly processes of the cytoskeleton, attenuate infammation, and induce immunomodulatory efect [31,32].It was claimed that the phytoantioxidants content in garlic extract could neutralize the hepatotoxicity of administrated heavy metals like lead nitrate and lead acetate [33,34].Because the treatment of aqueous garlic extract was able to reverse these oxidant reactions, as well as improve renal function and histological damage, it is plausible that aqueous garlic extract shields the tissues from nicotine-induced oxidative damage [35].Furthermore, both garlic oil and diallyl disulfde have shown promise as possible anti-infammatory agents in the treatment of airway infammation caused by cigarette smoking [36].Aged red garlic extract inhibits cisplatin-induced cell death in human bronchial smooth muscle cells by boosting reduced glutathione concentration and decreasing reactive oxygen species production [37].
Several studies have shown the protective efect of honey on other body tissues.Te administration of honey reduced the alveolar wall damage in mice brought on by cigarette smoking by diminishing the lumen of the alveolus that had undergone enlargement and infltration of infammatory cells [38].When chrysin (a polyphenol available in honey) was administered intraperitoneally in various dosages, it prevented cigarette smoke-induced infammation by decreasing the infammatory cytokines and myeloperoxidase activity by lowering the phosphorylation of p38 and ERK [39,40].Honey protects rat testes from smoking-induced histological alterations and oxidative stress, according to several studies [41,42].Honey considerably decreased the harmful efects of cigarette smoking on testicular structures and oxidative stress by lowering lipid peroxidation and restoring the antioxidant system [43,44].

Conclusion
Tis study proposes that the aqueous extract of honey and garlic may possess a mitigating efect on oxidative damage in the specifc organs examined in this research, which was induced by short-term subacute secondhand (passive) exposure to cigarette tobacco smoke.Currently, smoking cessation is widely regarded as the most efective approach to mitigate the detrimental efects of inhaled toxins.

Figure 1 :Figure 2 :
Figure1: Te concentration of two oxidative stress markers in the serum of experimental mice in this study was presented by a mean ± SEM plotting graph.(a) Concentration of protein carbonyl content in nmol/ml; (b) concentration of malondialdehyde in nmol/ml; signifcancy versus control group represented by ( * asterisks); signifcancy versus control group represented by (# hashtag).Te blue-capped lines represent the signifcance between the intergroups and could be assigned with $, $$, $$$, N.S. single, double, triple, and quadruple signs indicate P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively, and N.S represents no signifcance when P ≥ 0.05.

Figure 4 :
Figure 4: Images of jejunum sections for the experimental groups in this study stained with hematoxylin and eosin: (a) control group, (b) exposure to cigarette smoking, (c) exposure to cigarette smoking with parallel treatment to honey, and (d) exposure to cigarette smoking with parallel treatment to aqueous garlic extract.Tese photos were taken with a colored digital camera (Leica EC3, Switzerland) attached to a Leica inverted light microscope (Leica microsystems, Germany), and they were manually examined using computer software (Leica application suite LAS EZ version 1.8.0,Leica microsystems, Switzerland).

Figure 5 :
Figure 5: Ultrathin sections of 70 nm from livers of diferent experimental groups.(a) Control group, (b) cigarette smoking showing many vacuoles (arrows), (c) cigarette smoking + honey, and (d) cigarette smoking + garlic.Sections were stained with uranyl acetate and counterstained with lead citrate; the scale bars presented in the lower right corner of the images; these images were captured using a transmission electron microscope (Morgagni 268, FEI Philips, Netherlands), monitored through computer software (Morgagni 268D, version 2.21, Netherlands).

Figure 6 :
Figure 6: Ultrathin sections of 70 nm from kidney of diferent experimental groups.(a) Control group, (b) cigarette smoking, (c) cigarette smoking + honey, and (d) cigarette smoking + garlic.Sections were stained with uranyl acetate and counter-stained with lead citrate; the scale bars presented in the lower right corner of the images; these images were captured using transmission electron microscope (Morgagni 268, FEI Philips, Netherlands), monitored through computer software (Morgagni 268D, version 2.21, Netherlands).Te ultrathin sections were photographed at 60 kilovolts at diferent low power magnifcations.

Figure 7 :
Figure 7: Ultrathin sections of 70 nm from jejunum of diferent experimental groups.(a) Control group, (b) cigarette smoking, (c) cigarette smoking + honey, and (d) cigarette smoking + garlic.Sections were stained with uranyl acetate and counter-stained with lead citrate; the scale bars presented in the lower right corner of the images; these images were captured using a transmission electron microscope (Morgagni 268, FEI Philips, Netherlands), monitored through computer software (Morgagni 268D, version 2.21, Netherlands).Te ultrathin sections were photographed at 60 kilovolts at diferent low power magnifcations.